Apoptosis is a physiological process of cell death that is critical for normal development and tissue homeostasis. Defects in the regulation of apoptotic mechanisms contribute to the pathogenesis of multiple diseases, including those with reduced rates of apoptosis (cancer, autoimmunity) or with excessive cell death (neurodegeneration, stroke, myocardial infarction). The primary focus of this proposal is to elucidate the genetic mechanisms that regulate and execute cell death in the context of a developing organism. We are utilizing the highly accessible genetic model organism Drosophila melanogaster. In Drosophila, the basic components of the cell death machinery are conserved. Homologs of caspases, ced-4/Apaf-1, and lAPs have been identified. We have performed a genetic mutagenesis screen aimed at identifying mutants in components of the cell death machinery in Drosophila. These mutants are extremely informative for the genetic dissection of the Drosophila cell death pathway. For instance, genetic analysis of a subset of these mutants identified the Ras/MAPK pathway as important negative regulator of Hid, one of the cell death-inducing genes in flies. This finding is significant as 30% of human tumors are associated with oncogenic forms of Ras. Therefore, we devote two specific aims to analyze this interaction. We will determine the biochemical basis of Ras/MAPK-induced inhibition of Hid, and we will molecularly identify an additional gene, shes, that appears to control the MAPK/Hid interaction. Caspases, the principal effectors of apoptosis, are under tight genetic control, lAPs inhibit the activity of caspases, whereas Ced-4/Apaf-l-like proteins are required for their activation. How lAPs and Ced-4/Apaf-1- like proteins coordinate caspase activation is poorly understood. Using mutants of the Drosophila homologs of lAPs and Ced-4/Apaf-1 we will dissect the genetic requirement of these genes for the control of caspase activation. Finally, we propose a novel approach that will permit us to isolate additional as yet uncharacterized components of the Drosophila cell death pathway. The information obtained in these experiments will provide new insights into human diseases where deregulation of apoptosis is known to occur and may lead to new strategies for therapeutic intervention.